1. Field of the Invention
The present invention is generally related to the enantioselective production of amino acids and chiral amines using transaminase biocatalysts. More particularly, the present invention relates to the use of peroxides to increase the yield and improve purification of non-naturally occurring amino acids and chiral amines.
2. Description of the Prior Art
Amino acids, including those not found in nature, are of increasing industrial importance because of their applications as intermediates in the pharmaceutical and agrochemical industries. Chiral amines and non-naturally occurring amino acids are two of the most valuable and rapidly growing classes of chemical compounds used in pharmaceutical, chemical and agrochemical discovery and development. The high value of these compounds is partly due to the difficulty of manufacturing them on a large scale. Part of this difficulty arises because many valuable amino acids and amines exist in two distinct 3-dimensional forms in a mixture that is difficult to separate, and usually only one form is required for a particular application. Generally, but with the exception of glycine, each of the common proteinogenic amino acids has a chiral, or asymmetric, α-carbon since there are four different functional groups bonded to the α-carbon. Thus, amino acids can exist as stereoisomers, which are compounds with the same molecular formula that differ in arrangement or configuration of their atoms in space. Enantiomers are stereoisomers, which are non-superimposable mirror images, and can exist for each chiral amino acid. The mirror image pairs of amino acids are designated D, dextrorotary, or L, levorotary, depending on whether the α-carbon of the amino acid corresponds to the D- or L-enantiomer of glyceraldehyde, the common reference compound. Most naturally occurring amino acids are of the L-configuration, although a number of D-amino acids occur in nature. Similarly the enantiomers of a given chiral amine are designated S- or R-according to their particular properties of optical rotation.
Chirality is critical to the function of compounds. In many pharmaceutical applications, the FDA has mandated that only one enantiomer of a compound may be used in a particular drug, and the opposite enantiomer may not be present at all. Thus, chemical and physical methods have been sought to prepare or separate the individual enantiomers of an amino acid or amine. To meet the industrial demand for these compounds, many methods have been described and developed to prepare enantiomerically pure amino acids and amines. These methods include the physical separation or resolution of enantiomeric pairs using chromatographic or crystallization methods, biocatalytic resolution of enantiomers using enzymes, asymmetric synthesis of single enantiomers using chemo- or biological catalysts, and, particularly for natural amino acids, fermentation methods using engineered microbes. Although each of these approaches has noted advantages in specific instances, each has been limited by narrow applicability to a few specific amino acids or amines required by the industry, and many are inherently compromised by low efficiency and relatively low yields.
For example, fermentation methods are limited to the production of natural amino acids, whereas most of the amino acids required for pharmaceutical and agrochemical applications do not occur in nature and accordingly are not suited for the complex biochemical pathways that are used in fermentative methods of production. Another approach has been to chemically manufacture amino acids and amines as racemic mixtures containing both D- and L-forms, and subsequently removing or destroying the undesired enantiomer by chemical or physical means in a process called resolution. Resolution methods are limited in almost all cases to a maximum single pass product yield of 50%, thereby incurring costs and generating waste in the form of solvents and unreacted by-products. Asymmetric synthesis of amino acids and amines using chemical and biological catalysts is often compromised by many factors including the narrow substrate ranges of the chemo- and biocatalysts used, inaccessible or expensive starting materials and stringent operating parameters for the catalysts including the need for organic solvents, chemo-catalysts, or complex methods to contain and regenerate cofactors required for the biocatalysts. As a result of these limitations, more general and robust processes have been sought for the commercial preparation of amino acids and chiral amines.
One biocatalytic method that has proven both robust and general to prepare D- and L-amino acids (both natural and unnatural) with greater than 99.9% enantiomeric purity employs microbial amino acid transaminase biocatalysts. Transaminases are well known in the art and have many properties desirable in an industrial biocatalyst. They often accept a broad range of substrates (starting materials), can be isolated widely from microbial sources, are stable, easily produced in recombinant systems, highly active and readily scaled up using low cost fermentation protocols. Amino acid transaminases exist in two stereoselective classes, either as L-selective or D-selective biocatalysts and so by choosing the appropriate transaminase an L- or D-amino acid can be made with high enantiomeric purity. Transaminases catalyze the reversible interconversion of amino acids and keto acids. The reversibility of this reaction is highly relevant to the present invention.
A general transaminase reaction is shown in FIG. 1A. In this reaction, a keto acid, which is the precursor of the desired amino acid product, is reacted with an amino acid called the amino donor. The transaminase enzyme exchanges the amino group of the amino donor with the keto group of the keto acid. The reaction therefore results in a new pair of amino and keto acids; the desired amino acid product and a new keto acid which is a by-product.
Transaminases, especially those from microbial sources, have been described in industrial applications to prepare unnatural amino acids. These transaminases include aromatic, aspartate and branched chain transaminases of Escherichia coli. Transaminase enzymes are ubiquitous throughout nature and many have been described in microbes. However, one of the main drawbacks to the efficiency and cost-competitiveness of transaminase based industrial bioprocesses is the equilibrium of this reaction, which typically provides close to a 1:1 ratio of substrates and products, wherein the “reverse” reaction occurs at the same rate as the forward reaction. Therefore, if at the start of the reaction, the composition of the starting material comprises 50% keto acid substrate and 50% amino donor, as shown in FIG. 1A, then at the end of the reaction the composition of the mixture will typically comprise 25% keto acid substrate and 25% amino donor and additionally 25% amino acid product and 25% keto acid by-product, as shown in FIG. 1B. The yield of the desired amino acid from its keto acid is therefore only 50% and the final product is only 25% of a mixture of four different chemicals. This means that 50% of the keto acid substrate is unreacted and wasted, and the isolation and purification of the desired amino acid product is severely compromised by the presence of a large number of unwanted chemicals.
Therefore, methods have been sought to influence the reaction yield by displacing the normal reaction equilibrium. Reducing or eliminating the keto acid by-product during the reaction can achieve this. Such removal in situ has the effect of reducing the rate of the reverse reaction such that the forward reaction dominates so that more substrate is then converted to product. In principle, if all the keto acid by-product can be removed then the reverse reaction can no longer occur and the forward reaction can proceed towards a theoretical 100% yield of the desired amino acid product. An additional benefit to the high yield of product would be that the amino acid donor would be completely consumed in the reaction, such that the only amino acid present in the final mixture is the desired product. In such a case, the isolation and purification of the amino acid product would be greatly simplified and improved.
Methods have been described to eliminate the keto acid by-product from the transaminase reaction, however all of these methods require that i) additional biocatalysts be added to the reaction; and/or ii) the amino acid donor used is aspartic acid. The additional enzymes that must be added increase the process cost and complexity, and also require to be produced by fermentation. Such processes lead to the generation of further by-products in the reaction. The limitation to having to use aspartic acid is due to the fact that the keto acid which derives from aspartic acid, oxaloacetic acid, can be readily decomposed, for example by additional enzymes and removed from the reaction, unlike many other keto acids. However, aspartic acid is not an ideal amino acid donor for this process, as most transaminase enzymes do not react with it and it can also lead to the production of further by-products such as the amino acid L-alanine.
The present invention provides a method to completely remove keto acid by-products from a transaminase reaction using peroxides thereby increasing the yield and purification of the desired amino acid product. Furthermore, the present invention is cost effective, does not require additional biocatalysts and is not limited to the use of aspartic acid as the amino donor.